Eimeria falciformis secretes extracellular vesicles to modulate proinflammatory response during interaction with mouse intestinal epithelial cells

Background Protozoan parasite secretions can be triggered by various modified media and diverse physicochemical stressors. Equally, host-parasite interactions are known to co-opt the exchange and secretion of soluble biochemical components. Analysis of Eimeria falciformis sporozoite secretions in response to interaction with mouse intestinal epithelial cells (MIECs) may reveal parasite secretory motifs, protein composition and inflammatory activities of E. falciformis extracellular vesicles (EVs). Methods Eimeria falciformis sporozoites were allowed to interact with inactivated MIECs. Parasite secretions were separated into EV and vesicle-free (VF) fractions by discontinuous centrifugation and ultracentrifugation. Secreted EVs were purified in an iodixanol density gradient medium and the protein composition of both EV and VF fractions were analyzed by liquid chromatoraphy-tandem mass spectroscopy. The inflammatory activities of E. falciformis sporozoite EV on MIECs were then investigated. Results During the interaction of E. falciformis sporozoites with inactivated MIECs, the parasite secreted VF and vesicle-bound molecules. Eimeria falciformis vesicles are typical pathogenic protozoan EVs with a mean diameter of 264 ± 2 nm, and enclosed heat shock protein (Hsp) 70 as classical EV marker. Refractile body-associated aspartyl proteinase (or eimepsin), GAP45 and aminopeptidase were the main components of E. falciformis sporozoite EVs, while VF proteins include Hsp90, actin, Vps54 and kinases, among others. Proteomic data revealed that E. falciformis EV and VF proteins are aggregates of bioactive, antigenic and immunogenic molecules which act in concert for E. falciformis sporozoite motility, pathogenesis and survival. Moreover, in MIECs, E. falciformis EVs induced upregulation of gene expression and secretion of IL-1β, IL-6, IL-17, IL-18, MCP1 as well as pyroptosis-dependent caspase 11 and NLRP6 inflammasomes with the concomitant secretion of lactate dehydrogenase. Conclusions Eimeria falciformis sporozoite interaction with MIECs triggered the secretion of immunogenic and antigenic proteins. In addition, E. falciformis sporozoite EVs constitute parasite-associated molecular pattern that induced inflammatory response and cell death. This study offers additional insight in the secretion and protein composition of E. falciformis secretomes as well as the proinflammatory functions of E. falciformis sporozoite EVs. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-022-05364-x.


Background
Host-parasite interaction is a dynamic and phenomenal feature of parasitism. It co-opts parasite development and pathogenesis by means of cellular signaling, inflammation, cell death and, essentially, the excretion or secretion of molecules. In general, parasite secretions (or secretomes) are formed along several secretory pathways and released during the host-parasite interplay [1]. Previous studies have described and characterized the secretion of extracellular vesicles (EVs) and vesicle-free (VF) molecules from parasitic protozoa and helminths [2,3] as well as from parasite-infected host cells [4]. Similarly, there have been several studies on the secretion, characterization and proteomic profiling of Eimeria sporozoite proteins triggered by thermal, chemical and mechanical stressors [5]. In addition, VF molecules have been induced and characterized from protozoan parasites in various modified mediums and by physicochemical stressors [5,6]. Pathogenic protozoan EVs activated by chemical stressors and serum-starved media have also been characterized and profiled [7]. A few studies have also indicated that host-parasite interactions could induce the secretion of vesicle-bound and VF molecules. In this regard, the formation of parasite secretomes could occur prior to [8] or during protozoan parasite attachment or invasion of the host cell [3,[8][9][10], but the description of distinct parasite secretions during interactions with the host cell, and characterization of such parasite-derived secretomes have not been demonstrated.
Typically, Eimeria spp. are obligate intracellular apicomplexan parasites [16]. More specifically, Eimeria falciformis naturally parasitizes caecal epithelial cells of wild and laboratory mice, causing catarrhal enteritis, hemorrhage and epithelial sloughing [17,18]. The life-cycle of E. falciformis is similar to that of other coccidian parasites, with the sporozoites, after excystation from the sporocyst, actively migrate through the mice gut before invading the host intestinal epithelial cells (IECs) [17,18,25]. In general, parasitization of the host cells by apicomplexan parasites involves gliding motility, cellular attachment, invasion and spontaneous formation of excretory/ secretory products with which apicomplexans modulate host cell responses [10,[26][27][28]. However, E. falciformis sporozoite proteins secreted during interaction with the host IECs have not been described [5]. Also, the secretion of parasite-derived EVs has not been characterized in Eimeria spp. [7], nor have the inflammatory responses of host IECs, such as the secretion of cytokines and formation of inflammsome complexes, to Eimeria-derived EVs.

Parasite propagation
Parasite propagation was carried out as described earlier [17,29,30] with modification. In brief, 100 specific pathogen-free Balb/c mice aged between 6 and 8 weeks were raised in plastic cages placed in a pathogen-free room with adequate supply of food and sterile water. Each mouse was orally inoculated with 50 µl of 2 × 10 5 E. falciformis sporulated oocysts/ml phosphate-buffered saline (PBS). Oocysts shed in feces between 7-and 11-day postinfection were recovered from homogenized fecal solution, washed through a metal strainer and purified by salt flotation. Purified oocysts were washed and suspended as appropriate in 2.5% potassium dichromate followed by incubation at 28 °C and 120 rpm for approximately inflammatory response and cell death. This study offers additional insight in the secretion and protein composition of E. falciformis secretomes as well as the proinflammatory functions of E. falciformis sporozoite EVs.

Purification of E. falciformis sporozoites
Eimeria falciformis sporozoites were excystated and purified as earlier reported [31][32][33]. Briefly, E. falciformis sporulated oocysts were washed in PBS to remove potassium dichromate through discontinuous centrifugation. Purified oocysts were precipitated in 40 ml of antibiotic-antimycotic diluent (1 part dilutent per 100 ml) (Solarbio, Beijing China). Eimeria falciformis sporulated oocysts were then suspended in 5% (v/v) sodium hypochlorite for 20 min on ice with intermittent stirring, and the oocysts were washed in PBS. The hypochloritetreated sporulated oocysts were then vortexed with an equal volume of glass beads (diameter: 0.5 mm) for 3 min to release sporocysts and thereafter incubated with 0.75% (w/v) sodium taurodeoxycholate and 0.25% (w/v) trypsin in PBS for 1 h at 37 °C and 200 rmp for sporozoite excystation. The final purification step was vacuum filtration of the E. falciformis sporozoites [19,32].

Separation of E. falciformis sporozoite secretomes
A 350-ml sample of culture supernatant was harvested without disrupting the MIEC monolayer and centrifuged at 4 °C sequentially at: 1000 g, 5 min; 2000 g, 10 min; 5000 g, 20 min; 10,000 g, 30 min. The supernatant was then transferred into polycarbonate ultracentrifugation tubes (Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged at 120,000 g for 18 h in a Sorvall WX+ Ultra Series centrifuge (T-890 fixed angle rotor, k-factor 25.1; Thermo Fisher Scientific). The resulting supernatant from the ultracentrifugation was subjected to ultrafiltration through a 0.22-µm Corning sterile filter (Corning, Corning, NY, USA) at 4 °C under gravity, followed by 10-fold concentration using an Ultra 10 kDa cut-off membrane (Amicon Ultrafiltration System; Milli-poreSigma, Burlington, MA, USA) and stored at − 80 °C as the E. falciformis sporozoite VF fraction [31]. The cell culture supernatant from the NCs was prepared in the same manner.
The resulting pellets from the ultracentrifugation process were pooled, reconstituted in 8 ml PBS (pH 7.2) and resolved on discontinuous iodixanol gradients solutions prepared by diluting OptiPrep ™ density gradient (ODG) medium (Sigma-Aldrich) in 60% (v/v) aqueous iodixanol with sterilized 0.3 M sucrose/10 mM Tris, pH 7.2 [32,33]. The gradient was formed by adding 1.6 ml each of 40, 20, 10 and 5% iodixanol solution into a 9-ml polycarbonate tube (Thermo Fisher Scientific) and 1 ml of EfSEV suspension was overlaid and subjected to ultracentrifugation for 20 h at 120,000 g and 4 °C in a Sorvall WX+ Ultra Series centrifuge (T-890 fixed angle rotor, k-factor 25.1; Thermo Fisher Scientific). Seven gradient layers were collected, resuspended in PBS (pH 7.2) and washed twice for 4 h at 120,000 g and 4 °C. EfSEV pellets were pooled, concentrated in 400 µl of PBS and stored at − 80 °C. Likewise, NC pellets were collected and washed appropriately. The density of EfSEVs was measured from the corresponding ODG layer after appropriate dilution at an optical density of 244 nm [32,33] with a UV-Visible spectrophotometer (Biomates 3S; Thermo Fisher Scientific) (Additional file 6 : Data set).

Transmission electron microscopy
For transmission electron microscopy (TEM), 10 μl of EfSEVs was added to the a formvar carbon film-coated copper grid for 3 min. Excess liquid was removed with filter paper followed by staining with 3% phosphotungstic acid [34,35]. An equal volume of NC particles was also stained. The grids were observed in model HT770 transmission electron microscope (Hitachi, Tokyo, Japan).

Nanoparticle tracking analysis
The size of EfSEVs was determined using the Zetasizer Nano-Zs instrument (Malven, Worcestershire, UK). Briefly, 10 μg of EfSEVs and an equal volume of NC pellets were dissolved in an appropriate volume of PBS to make a total volume of 1 ml in each case. The size and distribution of the particles were measured in triplicate [13].

Sodium dodecyl sulfate-polyacryamide gel electrophoresis and liquid chromatography-tandem mass spectrometry
The protein components of the EfSEV and VF fractions were quantified using the BCA kit (Solarbio, Beijing, China). An equivalent of 10 µg of EfSEV and VF fractions, as well as the NC and NCs were separately mixed with lysis buffer (Solarbio, Beijing, China) according to the manufacturer's instruction, heated in a water bath at 100 °C for 10 min and resolved by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) [35,36] at 100 V in a Mini-Cell electrophoresis system (BioRad Laboratories, Hercules, CA, USA). Following electrophoresis, the SDS-PAGE gel was stained with a Coomassie blue stain kit. Subsequently, the EfSEV and VF protein gel lanes were excised and pooled for mass spectrometry (MS).
Liquid chromatography-tandem MS (LC-MS/MS) was performed by the Lu-Ming Biotech Co., Ltd. (Shanghai, China). In brief, excised protein gel lanes were dehydrated and dried in a vacuum. Gel particles were reduced in 10 mM DTT/25 mM NH 4 HCO 3 followed by treatment with alkylation buffer and recovered in 25 mM NH 4 HCO 3 . Reduced gel particles were digested in 20 μl of 0.02 μg/μl trypsin in 25 mM NH 4 HCO 3 . The supernatant was treated with 50 μl of 5% formic acid/67% acetonitrile followed by elution in 5% formic acid/67% ACN/ H 2 O. After drying using low-pressure centrifugation, the sample was re-dissolved in 40 µl of 0.1% formic acid and centrifuged through a column. The digested peptide elute was adjusted to pH 7 by H 3 PO 4 and the resulting peptides were lyophilized in 150 μl of 100% methanol. MS analyses were performed with a Q-Exactive mass spectrometer (Thermo Fisher Scientific) equipped with Nanospray Flex source (Thermo Fisher Scientific). MS/MS spectra were obtained with a resolution of 17, 500 and an AGC target of 2e 5 at a maximum injection time of 50 ms. The Q-E dynamic exclusion was set at 15.0 s in positive mode.

Protein database search and bioinformatics
MaxQuant, a quantitative proteomics software package, was used to search peptide sequences against Eimeria species, Toxoplasma gondii and Plasmodium falciparum proteins in UniProt. Trypsin digestion specificity was used to search databases. For protein quantification, MS1 and MS2 tolerance were set at 10 ppm and 0.02 Da, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analyses were also performed.

Western blot
EfSEV and VF protein concentrations were measured using a BCA kit (Solarbio, Beijing, China). An equivalent volume (10 µg) of EfSEV, VF, NC and NCs was separately mixed with 4× Loading Buffer (Solarbio, Beijing, China) and vortexed. The mixture was heated in a water bath at 100 °C for 10 min. The protein components were resolved in a 12% SDS-PAGE gel, and the protein bands were transferred to a polyvinylidene difluoride membrane (Merck-Millipore, Germany). After 1 h of blocking with 0.01% Tween-20 in PBS (PBST) containing 5% skimmed milk, the membrane was incubated with antiheat shock proteins 70 kDa (Hsp70; Proteintech Group Inc., Rosemont, IL, USA) overnight at 4 °C. Thereafter, the membrane was washed in PBST and incubated with horseradish protein-conjugated antibodies (Proteintech Group) for 1 h at room temperature. The membrane was treated with the WesternBright ™ ECL enhanced chemiluminescent substrate kit (Advansta Inc., San Jose, CA, USA) according to the manufacturer's instruction and visualized using the Luminescent Image Analyzer Amersham Imager 600 series (GE Co., Boston, MA, USA).

RNA extraction and quantitative PCR
RNA was extracted from EfSEV-stimulated MIECs using TRIzol reagents (Invitrogen, Thermo Fisher Scientific). In parallel, the same density of MIEC was treated with NC particles. A 1-µg aliquot of total RNA was reverse transcribed with the Primescript ™ RT Reagent Kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. PCR primers for IL-1β, IL-6, IL-17, IL-18, MCP1, caspase 11 and NLRP6 were synthesized by Tsingke Biological Technology (Xi'an, China) (Additional file 5). Real-time quantitative PCR (qPCR) was performed using TB Green pre-mix Ex Taq ™ II (Takara Bio Inc.) and the CFX98 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Each reaction tube contained 1 μl each of forward and reverse primers, 2 μl of cDNA template, 10 μl of TB Green and 6 μl of nucleasefree water. Assays were performed in triplicate, and data were normalized by using β-actin as a reference gene.

Lactate dehydrogenase assay
Approximately 1 × 10 6 MIECs in DMEM supplemented with 10% exosome-depleted FBS were stimulated with 50 µg of EfSEVs for 0, 6, 12, 18 and 24 h. Likewise, 10, 30, 50, and 100 µg of EfSEVs were used to stimulate MIECs and PBS (0 µg of EfSEVs) as experimental control treatment. Also, an equivalent volume of NC particles was used as a negative control for EfSEV time-and dosedependent production of lactate dehydrogenase (LDH) by MIECs [39,40]. The culture plates were incubated at 37 °C and 5% CO 2 . LDH was measured by Chekine ™ LDH assay kit (Abbkine, Wuhan, China) as instructed in the product manual. Absorbance was measured at 450 nm on a SpectraMax M5e Multi-Mode Microplate Reader (Molecular Devices, LLC, San Jose, CA, USA).

Statistical analyses
Data were expressed as the mean with the standard deviation and analyzed by one-way analysis of variance. Turkey's test was used for multiple comparisons. Linear regression analysis was performed using SPSS software version 21 (SPS IBM Corp., Armonk, NY, USA). Charts were generated in GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Differences among dose-and time-dependent activities of EfSEVs on MIECs relative to the experimental control were considered to be statistically significant at *P < 0.05, **P < 0.001 and ***P < 0.0001.

E. falciformis sporozoite secretomes during interaction with MIECs
The E. falciformis sporozoite (Fig. 1a) is the motile, invasive stage of the parasite which navigates through the host gut before invading the host cell. The fractions of E. falciformis sporozoite secretome were separated from the culture medium by serial centrifugation and ultracentrifugation steps, and then the scereted EVs were characterized by nanoparticle tracking analysis (NTA) and TEM. Also, EfSEV and VF protein compositions were identified by electrophoresis (Additional file 5) and analyzed by MS (Additional file 6). Our findings revealed that EfSEVs had a buoyant density ranging from 1.12 to 1.179 g/cm 3 distributed between the second and the seventh ODG layers. The particles from NC had a buoyant density that ranged between 0.57 and 1.103 g/cm 3 (Additional file 5). NTA analysis revealed that EfSEVs were a heterogeneous population of EVs with a diameter of approximately 37.84-1,281 nm (Fig. 1b). TEM observations indicated that EfSEVs were spherical and circular in shape, similar to Toxoplasma gondii and Neospora caninum EVs [35,41] (Fig. 1c). In addition, TEM revealed that NC particles (Additional file 2) did not contain secreted vesicles, indicating that the observed EVs were formed during E. falciformis sporozoite interaction with MIECs. Additionally, the vesicular property of EfSEVs and depletion of protein in the non-treated control experiment were also confirmed by targeting Hsp70 in EfSEVs, VF, NC, and NCs for western blotting (Fig. 1d).

Protein composition of E. falciformis sporozoite EV and VF fractions
Electrophoretic analyses of the EfSEV and VF fractions showed that E. falciformis sporozoites secreted proteins as components of its secretomes (Additional file 6) during interaction with inactivated MIECs. The protein composition of the EfSEV and VF fractions were analyzed by LC-MS/MS. MS/MS spectra were searched in the protein database for avian and bovine Eimeria species, T. gondii and P. falciparum because of the intractable genome of E. falciformis [9]. Forty-two proteins were identified from the E. falciformis sporozoite VF fraction (Additional file 6), including actin (putative), kinases, heat shock proteins (Hsp 70 and Hsp90), vacuolar protein sorting (Vps) 54, elongation factor 1 alpha (EF-1α), early gametocyte enriched phosphoprotein (EGXP), as well as some P. falciparum and T. gondii orthologous proteins ( Fig. 2; Additional file 6). GO terms for E. falciformis sporozoite VF protein are shown in Fig. 3a.
Thirty-seven proteins were identified from EfSEVs along with other unidentified protein peptides ( Fig. 2; Additional file 6). Identified EfSEV proteins include refractile body (RB)-associated proteins, Hsp70, EF-1α, cytosol aminopeptidase (putative), aspartyl proteinase (or eimepsin) and proteases. Other proteins from EfSEVs were gliding associated protein (GAP) 45, histones and some orthologous proteins from T. gondii and P. falciparum ( Fig. 2; Additional file 6). EfSEV proteins relating to biological processes, cell components and molecular functions are shown in Fig. 3. Also, the protein composition of EfSEV implies the involvement of these proteins in E. falciformis sporozoite binding/fusion to the host cell, endocytosis and metabolic processes (Fig. 3). KEGG analysis revealed that EfSEV proteins were enriched in pathways involving endocytosis, protein processing, RNA transport, spliceosome and ribosome, as found in N. caninum EV proteins [35], as well as the glutathione metabolism pathway, which is associated with gene expression, DNA and protein synthesis, signal transduction, cytokine production and immune responses ( Fig. 3; Additional file 5).

EfSEVs induced IL-1β, IL-6, IL-17, IL-18 and MCP1 production in MIECs
Intestinal epithelial cells are known to secret cytokines and chemokines as well as cellular infiltration during inflammatory responses and parasite-host interactions [17,42]. Also, Eimeria species can induce the secretion of diverse cytokines/chemokines in the host intestinal epithelium, including IL-6, IL-17, IL-18, IL-1β, interferon gamma (INFγ) [17,44] and MCP1 [43]. However, the reactive antigenicity of Eimeria-derived EVs in host inflammatory responses during infection is not fully understood. Therefore, we examined the secretion of MIEC proinflammatory cytokines after EfSEV stimulation. The results showed that MIECs produced quantifiable IL-1β, IL-18, IL-17 and IL-6 in response to EfSEV dose and time stimulations. While the level of IL-6 significantly increased with increasing time of MIEC stimulation by EfSEVs, the production of IL-17 reduced (Fig. 4). In addition, the production of IL-6 and IL-17 increased at high doses of EfSEVs (Fig. 4). Also, the production of MCP1 significantly increased with increasing EfSEV doses and time of stimulation (Fig. 4). The secretion of IL-18 by EfSEVs-stimulated MIECs was initially low but increased with time and with increasing doses of EfSEVs. Similarly, the production of IL-1β only increased significantly at a high dose of EfSEVs (Fig. 4). Taken together, EfSEV dose enhanced the production of proinflammatory cytokines in MIECs whereas the production of MCP1 was significantly increased with increasing time of stimulation and dosage of EfSEVs (Fig. 4).
Gene expression profiles of the selected cytokines and chemokine also indicated steady and significant  for 24 h. Also, 50 µg of EfSEVs were used to stimulate 1 × 10 6 MIECs for 6, 12, 18 and 0 h as experimental control. IL-6, IL-17, MCP1, IL-1β and IL-18 secretions in cell culture supernatants were measured by protein microarray kit. The signals of the laser scanning map were extracted with GenePix 6.0 microarray analysis software. Quantitative data obtained from the Quantibody-INF-1Q-Analyzer were analyzed using RayBiotech mouse Inflammation Array 1 software. Cytokine concentrations (pg/ml) were determined by mean fluorescence intensities and linear regression standard curves were generated from the manufacturer's standard. Each spot on the graph represents quantitative mean ± SD values in triplicate experiments. Asterisks indicate statistical significant differences from the control treatments at: *P < 0.05, **P < 0.001, ***P < 0.0001. Abbreviations: IL, Interleukin; MCPI, monocyte chemoattractant protein 1; MIECs, mouse intestinal epithelial cells; PBS, phosphate-buffered saline upregulation of MCP1 and IL-6 mRNA expression with increasing time of stimulation and EfSEV doses, but the increased IL-17 mRNA expression was just significant at high concentration of EfSEVs (Fig. 5). IL-18 mRNA expression was also significantly upregulated with increasing dose of EfSEVs and time of treatment (Fig. 5). However, mRNA expression of INFγ remained unchanged with increasing time and EfSEV doses (data not shown). In essence, EfSEVs are parasite components that modulate proinflammatory cytokines and chemokine genes during Eimeria-host interactions (Fig. 5). The apparent inconsequential change in the expression of the cytokines with NC particles indicated that the observed relative expressions of IL-6, IL-17, IL-18, IL-1β and MCP1 were due to the MIEC responses to EfSEV bioactivities (Fig. 5&6).

EfSEVs upregulated MIEC pyroptotic inflammasomes and induced the release of LDH
The eventual outcomes of pathogen-host interactions are sometimes marked by inflammation and host cell death [45]. Given the functional association of IL-18 and IL-1β with inflammatory programmed cell death, we investigated the regulatory activities of caspase 11 and NLRP6 in EfSEV-stimulated MIECs by qPCR. The analysis showed that increasing EfSEV dose and increasing duration of stimulation significantly upregulated the mRNA expression of NLRP6 and caspase 11 in MIECs whereas NC particles showed no distinct effects on inflammasome activation (Fig. 6a, b).
Cytosolic protein ligands, such as pathogen-associated molecular patterns (PAMPs), have been shown to activate pyroptosis-dependent IEC death. Specifically, pyroptosis of IECs can occur via the activation of caspase 11 and NLRP6 with a concomitant secretion of IL-18 and IL-1β [46] with the release of cytosolic LDH [39]. Therefore, LDH production by EfSEV-stimulated MIECs was investigated. The increased production of LDH by MIECs was significant from 18 h of EfSEV treatment and with increasing doses of EfSEVs. The result indicated that EfSEVs induced significant production of LDH as a marker for proinflammatory MIEC death. This finding was supported by the observation that NC particles showed no significant involvement in the production of LDH by MIECs (Fig. 6c).

Discussion
Previous studies have demonstrated that protozoan parasite secretomes include vesicle-bound and VF molecules such as nucleic acids, carbohydrates or glycocnjugates, lipids and proteins [6,7,47]. In this study, E. falciformis sporozoite EV and VF proteins were resolved from the total secretome formed while the parasite interacted with inactivated MIECs, and the protein components were analyzed by gel electrophoresis and LC-MS/MS. The electrophoretic profiles revealed protein bands in EfSEV and VF gel lanes, but no specific band was visible from NC and NCs, indicating that EfSEVs and VF preparations were free from MIEC secretions (Fig. 1d) (Additional file 5). This observation is in conformity with T. gondii tachyzoites which were unable to secret vesicles after being treated with aldehyde [48]. Notwithstanding, EfSEVs are subpopulations of self-assembled spherical and circular-shaped EVs with an expression of Hsp70 as membrane marker (Fig. 1c, d).
The proteomic dataset showed that EfSEVs and the VF fraction comprised membrane-associated and bioactive proteins (Additional file 6). Functionally, the gliding motility of apicomplexan parasites requires actin polymerization [49], and E. falciformis sporozoite VF Hsp90 might be involved in host cell invasion, host-parasite interaction, signal transduction and parasite development similarly as found in Eimeria tenella, T. gondii, and P. falciparum Hsp90 [50]. Also, the Vps51-54 complex has been suggested to mediate host endocytosis of T. gondii and P. falciparum [51,52]. Vps54 would possibly play a similar role during E. falciformis sporozoite invasion (Fig. 2). Additionally, Cryptosporidium parvum EF-1α has been associated with host cell invasion [53], and E. tenella protein kinases are immunogens [54] known to inhibit apoptosis and play major roles during stress [55,56]. Congruently, E. falciformis VF proteins are likely to be important for sporozoite motility, survival, invasion and pathogenesis.
Moreover, EF-1α and Hsp70 have been identified as EV markers of Leishmania major and other parasitic protozoa [57]. The identification of Hsp70 and EF-1α in EfSEV and VF proteins (Fig. 2) suggests that specific protein could be secreted via two or more secretory pathways during host-parasite interactions. The secretion of Hsp70 by Eimeria species is associated with sporozoite formation, pathogenicity, protective immunity and response to stress [5,58,59]. Hsp70 has also been reported among EV proteins from C. parvum, N. caninum, T. gondii, Leishmania and Trypanosoma species [7]. EfSEV proteases are likely to play important roles in E. falciformis sporozoite invasion, evasion of immune cells [60], developmental regulation and virulence, as revealed in other Apicomplexa [61], as well as degrading the cell matrix of host tissue [62]. Also, proteases are transcriptional regulators of E. tenella, Plasmodium and T. gondii life-cycles [5,63] and, more importantly, the invasion of Eimeria sporozoites is accompanied by proteolytic shedding of surface adhesins mediated by protease [64] (Fig. 2). h. An equivalent volume of PBS (0 µg of EfSEVs) was used as experimental control for the dose treatment and 0 h was used as control for the time-dependent treatment. An equal volume of NC particles was set as a negative control. RNAs were extracted and analyzed by qPCR for caspase 11 and NLRP6 mRNA expression. Bars represent mean ± standard deviation values in triplicate experiments. The difference in mRNA expression was compared with control treatments and considered to be significant at *P < 0.05, **P < 0.001, ***P < 0.001. c EfSEVs induced secretion of LDH in MIECs. 1 × 10 6 MIECs were stimulated with increasing doses (10, 20, 30, 50, 100 µg) of EfSEVs. Also, 50 µg of EfSEVs was used to stimulate MIECs at increasing duration (0, 6, 12, 18, 24 h). PBS (0 µg of EfSEVs) was used as experimental control for the dose treatment and 0 h as the control for time-dependent treatment. An equal volume of NC particles was set as a negative control in both cases. MIECs were harvested at the end of the treatment and assayed for LDH production using the Chekine ™ LDH assay kit in triplicate. Differences in the time-and dose-dependent release of LDH were compared with the control treatments and considered to be significant at *P < 0.05, **P < 0.001, ***P < 0.001. Abbreviations: LDH, Lactate dehydrogenase; NLRP6, nucleotide-oligomerization domain (NOD)-like receptor pyrin 6 Refractile body (RB)-derived functional molecules include a number of proteins/enzymes, such as aspartyl proteinase (eimepsin), RB antigen (SO7), and several haloacid dehalogenases such as hydrolase, subtilase, transhydrogenase and ubiquitin, as well as some carbohydrates [65,66]. Eimepsin is an Eimeria-specific antigenic protein [67,68] that is abundantly secreted in the sporozoite stage [5]. SO7 is also highly immunogenic with conserved antigenic epitopes among Eimeria species infecting chicken [69]. SO7 has been reported to effectively stimulate the immune response compared with single fragment vaccine [70], and it also plays important roles in host cell invasion, secretion of microneme proteins and Eimeria parasite intracellular survival [69]. In addition, the E. falciformis and E. tenella leucine aminopeptidase is an enzyme which preferentially catalyzes the hydrolysis of leucine residues at the N-termnus of peptides and proteins [71], which could be crucial for invasion, immune response, parasite excystment [72] and functions similar to the EV-bound aminopeptidase and serine protease of Acanthoamoeba castellanii [6,73]. Identification of EfSEV GAP45 suggests that the E. falciformis sporozoite has a trail of EVs during gliding motility [74,75] as reported in C. parvum infection [76]. Also, histones enclosed in EVs secreted by Leishmania donovani, P. falciparum, Trypanosoma brucei and T. cruzi could have functions in parasite viability and virulence [77][78][79], and EfSEV histones could have similar roles. Although EfSEV RNA content was low (data not shown), attending EfSEV histones suggest that E. falciformis EV cargo contained nucleic acids. Also, inactivation might have resulted in the loss of MIEC receptors [10] for E. falciformis sporozoite attachment, which in turn could have affected the number and composition of proteins in E. falciformis sporozoite secretomes.
IL-1β, IL-6, IL-17, IL-18 and MCP1 secretions are known to play crucial roles in E. tenella, C. parvum and T. gondii infections [80,81]. IEC IL-18 is constitutive and links innate and adaptive immunity during infection [82]. Similarly, MCP1 recruits monocytes and macrophages and co-regulates IL-1β in inflamed IECs [83]. Therefore, the expression of MCP1 (Figs. 4, 5) indicated that EfSEVs could play critical roles in the transcytosis of monocytes to the site of E. falciformis infection [21]. Also, the production of IL-1β could accelerate IL-17 response to PAMPs and stimulate chemokines recruiting leukocytes, T lymphocytes and neutrophils to the intestinal epithelium [84]. Then, E. falciformis sporozoites possibly secrete EVs to prevent early infiltration of cytotoxic CD4 + T cells to the site of infection [84] as MIEC IL-1β and IL-17 expressions were low in time-dependent responses to EfSEVs (Figs. 4,5) and it is comparable to the expressions of the IL-17 subset and neutrophils in T. cruzi-infected mice [85]. However, at a later time and high concentrations, EfSEVs induced significant expression of IL-17, as reported in mice infected with Leishmania major [86], suggesting that EfSEVs are potent, dose-dependent activators of inflammation and that the observed latent inflammatory effect of EfSEVs on MIEC might be associated with E. falciformis sporozoite behavior for intracellular survival.
It has been previously reported that Trichomonas vaginalis, Leishmania infantum, and P. falciparum EVs induced secretion of IL-6 from immune cells [7]. However, the secretion of IL-6 could be related to anti-inflammatory and anti-apoptotic responses in IECs [87,88]. It is possible that E. falciformis sporozoites secrete EVs to suppress MIEC apoptotic activities because it has been suggested that T. vaginalis exosomes upregulate IL-6 to suppress other proinflammatory cytokines [89]. This is in contrast to extracellular acidosis that has been reported to transiently modulate IL-6 expression in rat epithelial cell [90]. Nevertheless, EfSEVs enclosed potential danger/damage/stress-associated ligands that activated the release of endogenous IL-18 and IL-1β as proinflammatory cytokines associated with inflammasome complexes [91] (Figs. 5, 6). Also, EfSEVs [35,41] were able to bind green fluorescent dye and aggregate around the MIEC membrane with some degree of internalization at the later experimental time, as well as involvemnet in MIEC membrane disruption or sphericalization (Additional file 5). The cytoplasmic incorporation of EfSEVs by MIECs might render them as cytosolic ligands that subsequently activate inflammasome complexes [92].
Inflammasomes are vital innate immune complexes in protozoa-host interactions [93] and are activated by intracellular pattern-recognition receptors (or sensors) that recognize pathogen-danger- [94] and stress-associated ligands [95]. Inflammasomes include NLRPs, AIM2 and caspases [93,96]. NLRP inflammasome families contribute to intestinal disease pathogenesis while caspases participate in proinflammatory processes and pathophysiology of enteric pathogens [20,97,98]. In the present study, upregulation of MIEC caspase 11 and NLRP6, as well as dose-dependent upregulation of IL-18 and IL-1β (Figs. 5, 6) are comparable with inflammatory cascades and NLRP6 activation during mice colonic inflammation and tumorigenesis [46,96]. Likewise, the activation of NLRP6 during Cryptosporidium tyzzeri infection in mice caused a luminal release of IL-18 [99,100]. It is therefore not unlikely that C. tyzzeri-derived EVs, though not characterized, might have played pivotal role in the activation of NLRP6 in C. tyzzeri-infected mice, as observed in this study (Fig. 6a, b). Mechanistically, non-canonical caspase 11 is activated when toxins (e.g. bacterial lipopolysaccharide [LPS]) find their way into the host cell cytoplasm [97,101]. In the absence of the bacteria, Brucella melitensis LPS induced caspase 11 in murine enterocytes [102] like EfSEVs in this study. Moreover, the production of IL-1β, IL-18 and caspase 11 have been posited as markers for intestinal inflammation in response to pathogens [92,103].
Additionally, caspase 11-dependent pyroptosis, in association with IL-18, is a particularly important host immune mechanism against intracellular pathogens [92,102]. Also, the production of cytosolic LDH has been defined as a marker for inflammasome-dependent pyroptosis [98,104,105]. Upregulation of caspase 11 and NLRP6 with a significant release of LDH as well as dose-and time-dependent secretion of IL-1β and IL-18 [106] signified that EfSEVs are potential activators of pyroptosis in MIECs (Figs. 5, 6). Comparatively, the MIEC inflammatory response and the release of the cell death signal induced by EfSEVs were synchronous with the time of E. falciformis sporozoite intracellular egress [30]. However, the mechanism by which intracellular parasites secrete a regulated amount of EVs to temporarily delay host responses until the completion of intracellular development requires further investigation. It is therefore possible that inflammatory responses and host cell death during Eimeria infection are not entirely passive due to intraepithelial distress, but are prefigured by Eimeria parasite behavior for survival and successful completion of the life-cycle.

Conclusion
This study emphasized host-parasite interaction as an additional mechanism by which the protozoan parasite E. falciformis secretes EVs and VF molecules. The study unveils the protein compositions of E. falciformis sporozoite secretome as well as the MIEC inflammatory response to E. falciformis sporozoite EVs. Specifically, E. falciformis sporozoite EVs contained important antigenic and immunogenic proteins as well as other apicomplexan proteins that are implicated in motility, invasion, pathogenesis, survival and host cell degradation. Also, E. falciformis VF proteins encompassed proteins involved in pathogenesis, motility and vesicle formation. Furthermore, E. falciformis sporozoite EVs induced the release of proinflammatory molecules such as IL-6, IL-17, IL-1β and IL-18 as well as the upregulation of pyroptosis-dependent caspase 11 and NLRP6, at the least, in a dose-dependent manner. Essentially, E. falciformis EVs are components of danger-associated molecular patterns (DAMPs) with protein-and generegulatory activities that support pyroptosis-dependent response. Unlike parasite protein characterization through extraneous stimuli, the model of the study could be adapted to ascertain secretory motifs and constitutive antigens/immunogens in Eimeria species for the development of vaccines against poultry coccidiosis. Also, it will benefit our understanding to further describe the classical components of NLRP6-and caspase 11-mediated pyroptosis during E. falciformis infection.